U.S. patent application number 12/278354 was filed with the patent office on 2009-01-22 for polypeptides having organophosphorus hydrolase activity and polynucleotides encoding same.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Steffen Danielsen, Soeren Flensted Lassen, Lars Henrik Oestergaard.
Application Number | 20090025107 12/278354 |
Document ID | / |
Family ID | 36940609 |
Filed Date | 2009-01-22 |
United States Patent
Application |
20090025107 |
Kind Code |
A1 |
Oestergaard; Lars Henrik ;
et al. |
January 22, 2009 |
Polypeptides Having Organophosphorus Hydrolase Activity and
Polynucleotides Encoding Same
Abstract
The present invention relates to isolated polypeptides having
organophosphorus hydrolase (OPH) activity and isolated
polynucleotides encoding the polypeptides. The invention also
relates to nucleic acid constructs, vectors, and host cells
comprising the polynucleotides as well as methods for producing and
using the polypeptides.
Inventors: |
Oestergaard; Lars Henrik;
(Charlottenlund, DK) ; Lassen; Soeren Flensted;
(Farum, DK) ; Danielsen; Steffen; (Copenhagen,
DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE, SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
36940609 |
Appl. No.: |
12/278354 |
Filed: |
February 6, 2007 |
PCT Filed: |
February 6, 2007 |
PCT NO: |
PCT/DK07/00061 |
371 Date: |
August 5, 2008 |
Current U.S.
Class: |
800/295 ;
435/196; 435/252.3; 435/320.1; 435/410; 536/23.1; 536/23.2 |
Current CPC
Class: |
C12N 9/16 20130101 |
Class at
Publication: |
800/295 ;
435/196; 536/23.2; 435/252.3; 536/23.1; 435/410; 435/320.1 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C12N 9/16 20060101 C12N009/16; C07H 21/00 20060101
C07H021/00; C12N 15/64 20060101 C12N015/64; C12N 1/21 20060101
C12N001/21; C12N 5/04 20060101 C12N005/04 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 6, 2006 |
DK |
PA 2006 00159 |
Claims
1. An isolated polypeptide having organophosphatase activity,
selected from the group consisting of: (a) a polypeptide comprising
an amino acid sequence which has at least 87% identity with amino
acids 36 to 334 of SEQ ID NO: 2; (b) a polypeptide which is encoded
by a polynucleotide which hybridizes under at least medium
stringency conditions with (i) nucleotides 106 to 1002 of SEQ ID
NO: 1, or (ii) a complementary strand of (i); (c) a variant
comprising a conservative substitution, deletion, and/or insertion
of one or more amino acids of amino acids 36 to 334 of SEQ ID NO:
2.
2. The polypeptide of claim 1, comprising an amino acid sequence
which has at least 88% identity with amino acids 36 to 334 of SEQ
ID NO: 2.
3. The polypeptide of claim 2, comprising an amino acid sequence
which has at least 90% identity with amino acids 36 to 334 of SEQ
ID NO: 2.
4. The polypeptide of claim 3, comprising an amino acid sequence
which has at least 95% identity with amino acids 36 to 334 of SEQ
ID NO: 2.
5. An isolated polypeptide having organophosphatase activity
comprising the amino acids 36 to 334 of SEQ ID NO: 2.
6. The polypeptide of claim 1, which consists of amino acids 35 to
334 of SEQ ID NO: 2 or a fragment thereof having organophosphatase
activity.
7. An isolated polypeptide having organophosphatase activity, which
consists of SEQ ID NO: 2.
8. The polypeptide of claim 1, which is encoded by the
polynucleotide contained in plasmid pOPHNN10787 which is contained
in E. coli DSM 17765.
9. The isolated polypeptide according to claim 1, comprising the
cofactor Mn.
10. An isolated polynucleotide encoding an organophosphatase,
selected from the group consisting of: (a) a nucleic acid sequence
encoding a polypeptide comprising an amino acid sequence which has
at least 87% identity with amino acids 36 to 334 of SEQ ID NO: 2;
(b) a nucleic acid sequence having at least 81% identity with the
nucleic acid sequence from position 106 to 1002 of SEQ ID NO: 1;
(c) a nucleic acid sequence which hybridizes under medium
stringency conditions with (i) the nucleic acid sequence from
position 106 to 1002 of SEQ ID NO: 1, (ii) a subsequence of (i) of
at least 100 nucleotides, or (iii) a complementary strand of (i) or
(ii); (d) an allelic variant of (a), (b), (c); (e) a subsequence of
(a), (b), (c), or (d), wherein the subsequence encodes a
polypeptide fragment which has organophosphatase activity.
11. The isolated polynucleotide of claim 10, selected from the
group consisting of: (a) a nucleic acid sequences encoding a
polypeptide comprising the amino acid sequence of SEQ ID NO: 2; (b)
has the nucleic acid sequence of SEQ ID NO: 1; or (c) is contained
in plasmid pOPHNN10787 which is contained in E. coli DSM 17765.
12. The isolated polynucleotide of claim 10, having at least one
mutation in the mature polypeptide coding sequence of SEQ ID NO: 1,
in which the mutant nucleotide sequence encodes a polypeptide
consisting of amino acids 36 to 334 of SEQ ID NO: 2.
13. A nucleic acid construct comprising the polynucleotide of claim
10, operably linked to one or more control sequences that direct
the production of the polypeptide in an expression host.
14. A recombinant expression vector comprising the nucleic acid
construct of claim 13.
15. A recombinant host cell comprising the nucleic acid construct
of claim 13.
16. A method for producing the polypeptide of claim 1, comprising
(a) cultivating a cell, which in its wild-type form is capable of
producing the polypeptide, under conditions conducive for
production of the polypeptide; and (b) recovering the
polypeptide.
17. A method for producing the polypeptide of claim 1, comprising
(a) cultivating a host cell comprising a nucleic acid construct
comprising a nucleotide sequence encoding the polypeptide under
conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide.
18. A method for producing a polynucleotide having a mutant
nucleotide sequence, comprising (a) introducing at least one
mutation into the mature polypeptide coding sequence of SEQ ID NO:
1, wherein the mutant nucleotide sequence encodes a polypeptide at
least comprising amino acids 36 to 334 of SEQ ID NO: 2; and (b)
recovering the polynucleotide comprising the mutant nucleotide
sequence.
19. A method for producing the polypeptide of any of claim 1,
comprising (a) cultivating a transgenic plant or a plant cell
comprising a polynucleotide encoding a polypeptide having
organophosphatase activity of the present invention under
conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide.
20. A method for producing the polypeptide of claim 1, comprising
(a) cultivating an algae comprising a polynucleotide encoding a
polypeptide having organophosphatase activity of the present
invention under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
21. A use of the polypeptide according to claim 1, for hydrolysing
an organophosphorus molecule.
22. A composition for hydrolysing an organophosphorus molecule,
said composition comprising a polypeptide according to claim 1, and
one or more acceptable carriers.
23. A composition for hydrolysing an organophosphorus molecule,
said composition comprising a host cell according to claim 15.
24. A transgenic plant which produces a polypeptide according to
claim 1.
25. A method for hydrolysing an organophosphorus molecule, said
method comprising exposing the organophosphorus molecule to the
polypeptide according to claim 1.
Description
SEQUENCE LISTING
[0001] This application contains a Sequence Listing in computer
readable form. The computer readable form is incorporated herein by
reference.
DEPOSIT OF BIOLOGICAL MATERIAL
[0002] The following biological material has been deposited under
the terms of the Budapest Treaty with the DSMZ-Deutsche Sammiung
von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b,
D-38124 Braunschweig and given the following accession number:
TABLE-US-00001 Deposit Accession Number Date of Deposit Escherichia
coli DSM 17765 5 Dec. 2005
[0003] The strain has been deposited under conditions that assure
that access to the culture will be available during the pendency of
this patent application to one determined by the Commissioner of
Patents and Trademarks to be entitled thereto under 37 C.F.R.
.sctn.1.14 and 35 U.S.C. .sctn.122. The deposit represents a
substantially pure culture of the deposited strain. The deposit is
available as required by foreign patent laws in countries wherein
counterparts of the subject application or its progeny are filed.
However, it should be understood that the availability of a deposit
does not constitute a license to practice the subject invention in
derogation of patent rights granted by governmental action.
FIELD OF THE INVENTION
[0004] The present invention relates to isolated polypeptides
having organophosphorus hydrolase activity and isolated
polynucleotides encoding the polypeptides. The invention also
relates to nucleic acid constructs, vectors, and host cells
comprising the polynucleotides as well as methods for producing and
using the polypeptides.
BACKGROUND OF THE INVENTION
[0005] Organophosphates belong to a group of chemicals which
includes nerve agents, insecticidal and fungicidal agents. These
agents are highly toxic and thus means for detoxifying such agents
are highly desirable. Poisoning with organophosphates presents a
problem in agriculture as well as for military personnel exposed to
organophosphates used in chemical warfare. One way of detoxifying
such agents involves enzymatic degradation of organophosphates.
Enzymes capable of hydrolyzing organophosphates detoxify a variety
of nerve agents as well as commonly used pesticides.
Organophosphorus hydrolase (OPH) has been widely studied for its
activity on organophosphorus (OP) pesticides and its ability to
hydrolyse nerve agents. Suitable enzymes for degrading
organophosphate residues include OP hydrolases from bacteria
(Harper et al., 1988, Applied and Environmental Microbiology 54:
2586-2589; Mulbury and Karns, 1989, J. Bacteriol. 171: 6740-6746;
Mulbury, 1992, Gene 121: 149-153; Mulbury and Kearney, 1991, Crop
Protection 10: 334-345; Cheng et al., 1999, Chemico-Biological
Interactions 119-120:455-462; Horne et al., 2002, Applied and
Enveronmental Microbiology 68: 3371-3376; U.S. Pat. No. 5,484,728;
U.S. Pat. No. 5,589,386), vertebrates (Wang et al. 1998, J.
Biochem. and Mol. Toxicology 12: 213-217) and OP resistant insects
(WO 95/19440 and WO97/19176). It is desirable to find alternative
and/or improved OP hydrolyzing enzymes that can conveniently be
produced in sufficient amounts.
[0006] It is an object of the present invention to provide
polypeptides having organophosphatase activity and polynucleotides
encoding the polypeptides. Also such organophosphatase enzymes
should readily be expressed and purified from a fungal expression
system.
SUMMARY OF THE INVENTION
[0007] The present invention relates in a first aspect to isolated
polypeptide having organophosphatase activity, selected from the
group consisting of:
[0008] (a) a polypeptide comprising an amino acid sequence which
has at least 87% identity with amino acids 36 to 334 of SEQ ID NO:
2;
[0009] (b) a polypeptide which is encoded by a polynucleotide which
hybridizes under at least medium stringency conditions with (i)
nucleotides 106 to 1002 of SEQ ID NO: 1, or (ii) a complementary
strand of (i);
[0010] (c) a variant comprising a conservative substitution,
deletion, and/or insertion of one or more amino acids of amino
acids 36 to 334 of SEQ ID NO: 2;
In a second aspect the present invention relates to an isolated
polynucleotide encoding an organophosphatase, selected from the
group consisting of:
[0011] (a) a nucleic acid sequence encoding a polypeptide
comprising an amino acid sequence which has at least 87% identity
with amino acids 36 to 334 of SEQ ID NO: 2;
[0012] (b) a nucleic acid sequence having at least 81% identity
with the nucleic acid sequence 106 to 1002 of SEQ ID NO: 1;
[0013] (c) a nucleic acid sequence which hybridizes under medium
stringency conditions with (i) the nucleic acid sequence from
position 106 to 1002 of SEQ ID NO: 1, (ii) a subsequence of (i) of
at least 100 nucleotides, or (iii) a complementary strand of (i) or
(ii);
[0014] (d) an allelic variant of (a), (b), (c);
[0015] (e) a subsequence of (a), (b), (c), or (d), wherein the
subsequence encodes a polypeptide fragment which has
organophosphatase activity.
[0016] The present invention also relates to nucleic acid
constructs, recombinant expression vectors, and recombinant host
cells comprising polynucleotides encoding the polypeptides of the
invention.
[0017] The present invention also relates to methods for producing
such polypeptides having OPH activity comprising (a) cultivating a
host cell comprising a nucleic acid construct comprising a
polynucleotide encoding the polypeptide under conditions conducive
for production of the polypeptide; and (b) recovering the
polypeptide.
[0018] In a further aspect the present invention relates to a
method for producing a polynucleotide having a mutant nucleotide
sequence, comprising (a) introducing at least one mutation into the
mature polypeptide coding sequence of SEQ ID NO: 1, wherein the
mutant nucleotide sequence encodes a polypeptide comprising amino
acids 36 to 334 of SEQ ID NO: 2; and (b) recovering the
polynucleotide comprising the mutant nucleotide sequence.
[0019] In a still further aspect the present invention relates to a
method for producing the polypeptide of the invetnion, comprising
(a) cultivating a transgenic plant or a plant cell comprising a
polynucleotide encoding a polypeptide having organophosphatase
activity of the present invention under conditions conducive for
production of the polypeptide; and (b) recovering the
polypeptide.
[0020] In an even further aspect the present invention relates to a
method for producing the polypeptide of the invention, comprising
(a) cultivating an algae comprising a polynucleotide encoding a
polypeptide having organophosphatase activity of the present
invention under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
[0021] The present invention also relates to a use of the
polypeptide according to the invention for hydrolysing an
organophosphorus molecule.
[0022] In a still further aspect the invention relates to a
composition for hydrolysing an organophosphatase molecule, said
composition comprising a polypeptide according to the invention and
one or more acceptable carriers.
[0023] In another aspect the invention relates to a transgenic
plant which produces a polypeptide according to the invention.
[0024] Another aspect relates to a method for hydrolysing an
organophosphatase molecule, said method comprising exposing the
organophosphatase molecule to the polypeptide according to the
invention or the composition according to the invention or the
transgenic plant according to the invention.
DEFINITIONS
[0025] Organophosphatase activity: The term "organophosphatase
activity" is defined herein as an organophosphorus hydrolase (OPH)
(EC 3.1.8.1) activity which catalyzes the hydrolysis of
organophosphorus compounds. These enzymes are also known as
aryldialkylphosphatases. OPH's act on organophosphorus compounds,
such as paraoxin, including esters of phosphonic and phosphinic
acids. For purposes of the present invention, organophosphatse
activity is determined according to the procedure described in
EnzChek.RTM.Paraoxonase assay kit available from Molecular
Probes.TM.. An alternative assay for determining OPH activity is
described in Cho et al., 2004, Applied and Environmental
Microbiology, vol. 70(8):4681-4685.
[0026] The polypeptides of the present invention have at least 20%,
preferably at least 40%, more preferably at least 50%, more
preferably at least 60%, more preferably at least 70%, more
preferably at least 80%, even more preferably at least 90%, most
preferably at least 95%, and even most preferably at least 100% of
the organophosphatase activity of the polypeptide comprising the
amino acid sequence shown as amino acids 36 to 334 of SEQ ID NO:
2.
[0027] Isolated polypeptide: The term "isolated polypeptide" as
used herein refers to a polypeptide which is at least 20% pure,
preferably at least 40% pure, more preferably at least 60% pure,
even more preferably at least 80% pure, most preferably at least
90% pure, and even most preferably at least 95% pure, as determined
by SDS-PAGE.
[0028] Substantially pure polypeptide: The term "substantially pure
polypeptide" denotes herein a polypeptide preparation which
contains at most 10%, preferably at most 8%, more preferably at
most 6%, more preferably at most 5%, more preferably at most 4%, at
most 3%, even more preferably at most 2%, most preferably at most
1%, and even most preferably at most 0.5% by weight of other
polypeptide material with which it is natively associated. It is,
therefore, preferred that the substantially pure polypeptide is at
least 92% pure, preferably at least 94% pure, more preferably at
least 95% pure, more preferably at least 96% pure, more preferably
at least 96% pure, more preferably at least 97% pure, more
preferably at least 98% pure, even more preferably at least 99%,
most preferably at least 99.5% pure, and even most preferably 100%
pure by weight of the total polypeptide material present in the
preparation.
[0029] The polypeptides of the present invention are preferably in
a substantially pure form. In particular, it is preferred that the
polypeptides are in "essentially pure form", ie., that the
polypeptide preparation is essentially free of other polypeptide
material with which it is natively associated. This can be
accomplished, for example, by preparing the polypeptide by means of
well-known recombinant methods or by classical purification
methods.
[0030] Herein, the term "substantially pure polypeptide" is
synonymous with the terms "isolated polypeptide" and "polypeptide
in isolated form."
[0031] Identity: The relatedness between two amino acid sequences
is described by the parameter "identity".
[0032] For purposes of the present invention, the alignment of two
amino acid sequences is determined by using the Needle program from
the EMBOSS package (http://emboss.org) version 2.8.0. The Needle
program implements the global alignment algorithm described in
Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48,
443-453. The substitution matrix used is BLOSUM62, gap opening
penalty is 10, and gap extension penalty is 0.5.
[0033] The degree of identity between an amino acid sequence of the
present invention ("invention sequence"); e.g. amino acids 36 to
334 of SEQ ID NO: 2 and a different amino acid sequence ("foreign
sequence") is calculated as the number of exact matches in an
alignment of the two sequences, divided by the length of the
"invention sequence" or the length of the "foreign sequence",
whichever is the shortest. The result is expressed in percent
identity.
[0034] An exact match occurs when the "invention sequence" and the
"foreign sequence" have identical amino acid residues in the same
positions of the overlap (in the alignment example below this is
represented by "|"). The length of a sequence is the number of
amino acid residues in the sequence.
[0035] In the purely hypothetical alignment example below, the
overlap is the amino acid sequence "HTWGER-NL" of Sequence 1; or
the amino acid sequence "HGWGEDANL" of Sequence 2. In the example a
gap is indicated by a "-".
Hypothetical Alignment Example:
##STR00001##
[0037] In a particular embodiment, the percentage of identity of an
amino acid sequence of a polypeptide with, or to, amino acids 36 to
334 of SEQ ID NO: 2 is determined by i) aligning the two amino acid
sequences using the Needle program, with the BLOSUM62 substitution
matrix, a gap opening penalty of 10, and a gap extension penalty of
0.5; ii) counting the number of exact matches in the alignment;
iii) dividing the number of exact matches by the length of the
shortest of the two amino acid sequences, and iv) converting the
result of the division of iii) into percentage. The percentage of
identity to, or with, other sequences of the invention such as
amino acids 1-334 of SEQ ID NO: 2 are calculated in an analogous
way.
[0038] For purposes of the present invention, the degree of
identity between two nucleotide sequences is determined by the
Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the
National Academy of Science USA 80: 726-730) using the
LASERGENE.TM. MEGALIGN.TM. software (DNASTAR, Inc., Madison, Wis.)
with an identity table and the following multiple alignment
parameters: Gap penalty of 10 and gap length penalty of 10.
Pairwise alignment parameters are Ktuple=3, gap penalty=3, and
windows=20.
[0039] Polypeptide Fragment: The term "polypeptide fragment" is
defined herein as a polypeptide having one or more amino acids
deleted from the amino and/or carboxyl terminus of SEQ ID NO: 2 or
a homologous sequence thereof, wherein the fragment has OPH
activity.
[0040] Subsequence: The term "subsequence" is defined herein as a
nucleotide sequence having one or more nucleotides deleted from the
5' and/or 3' end of SEQ ID NO: 1 or a homologous sequence thereof,
wherein the subsequence encodes a polypeptide fragment having OPH
activity.
[0041] Allelic variant: The term "allelic variant" denotes herein
any of two or more alternative forms of a gene occupying the same
chromosomal locus. Alletic variation arises naturally through
mutation, and may result in polymorphism within populations. Gene
mutations can be silent (no change in the encoded polypeptide) or
may encode polypeptides having altered amino acid sequences. An
allelic variant of a polypeptide is a polypeptide encoded by an
allelic variant of a gene.
[0042] Substantially pure polynucleotide: The term "substantially
pure polynucleotide" as used herein refers to a polynucleotide
preparation free of other extraneous or unwanted nucleotides and in
a form suitable for use within genetically engineered protein
production systems. Thus, a substantially pure polynucleotide
contains at most 10%, preferably at most 8%, more preferably at
most 6%, more preferably at most 5%, more preferably at most 4%,
more preferably at most 3%, even more preferably at most 2%, most
preferably at most 1%, and even most preferably at most 0.5% by
weight of other polynucleotide material with which it is natively
associated. A substantially pure polynucleotide may, however,
include naturally occurring 5' and 3' untranslated regions, such as
promoters and terminators. It is preferred that the substantially
pure polynucleotide is at least 90% pure, preferably at least 92%
pure, more preferably at least 94% pure, more preferably at least
95% pure, more preferably at least 96% pure, more preferably at
least 97% pure, even more preferably at least 98% pure, most
preferably at least 99%, and even most preferably at least 99.5%
pure by weight. The polynucleotides of the present invention are
preferably in a substantially pure form. In particular, it is
preferred that the polynucleotides disclosed herein are in
"essentially pure form", i.e., that the polynucleotide preparation
is essentially free of other polynucleotide material with which it
is natively associated. Herein, the term "substantially pure
polynucleotide" is synonymous with the terms "isolated
polynucleotide" and "polynucleotide in isolated form." The
polynucleotides may be of genomic, cDNA, RNA, semisynthetic,
synthetic origin, or any combinations thereof.
[0043] Nucleic acid construct: The term "nucleic acid construct" as
used herein refers to a nucleic acid molecule, either single- or
double-stranded, which is isolated from a naturally occurring gene
or which is modified to contain segments of nucleic acids in a
manner that would not otherwise exist in nature. The term nucleic
acid construct is synonymous with the term "expression cassette"
when the nucleic acid construct contains the control sequences
required for expression of a coding sequence of the present
invention.
[0044] Control sequence: The term "control sequences" is defined
herein to include all components, which are necessary or
advantageous for the expression of a polynucleotide encoding a
polypeptide of the present invention. Each control sequence may be
native or foreign to the nucleotide sequence encoding the
polypeptide. Such control sequences include, but are not limited
to, a leader, polyadenylation sequence, propeptide sequence,
promoter, signal peptide sequence, and transcription terminator. At
a minimum, the control sequences include a promoter, and
transcriptional and translational stop signals. The control
sequences may be provided with linkers for the purpose of
introducing specific restriction sites facilitating ligation of the
control sequences with the coding region of the nucleotide sequence
encoding a polypeptide.
[0045] Operably linked: The term "operably linked" denotes herein a
configuration in which a control sequence is placed at an
appropriate position relative to the coding sequence of the
polynucleotide sequence such that the control sequence directs the
expression of the coding sequence of a polypeptide.
[0046] Coding sequence: When used herein the term "coding sequence"
means a nucleotide sequence, which directly specifies the amino
acid sequence of its protein product. The boundaries of the coding
sequence are generally determined by an open reading frame, which
usually begins with the ATG start codon or alternative start codons
such as GTG and TTG. The coding sequence may a DNA, cDNA, or
recombinant nucleotide sequence.
[0047] Expression: The term "expression" includes any step involved
in the production of the polypeptide including, but not limited to,
transcription, post-transcriptional modification, translation,
post-translational modification, and secretion.
[0048] Expression vector: The term "expression vector" is defined
herein as a linear or circular DNA molecule that comprises a
polynucleotide encoding a polypeptide of the invention, and which
is operably linked to additional nucleotides that provide for its
expression.
[0049] Host cell: The term "host cell", as used herein, includes
any cell type which is susceptible to transformation, transfection,
transduction, and the like with a nucleic acid construct comprising
a polynucleotide of the present invention.
[0050] Modification: The term "modification" means herein any
chemical modification of the polypeptide consisting of the amino
acids 1 to 334 of SEQ ID NO: 2 as well as genetic manipulation of
the DNA encoding that polypeptide. The modification(s) can be
substitution(s), deletion(s) and/or insertions(s) of the amino
acid(s) as well as replacement(s) of amino acid side chain(s).
[0051] Artificial variant: When used herein, the term "artificial
variant" means a polypeptide having OPH activity produced by an
organism expressing a modified nucleotide sequence of SEQ ID NO: 1.
The modified nucleotide sequence is obtained through human
intervention by modification of the nucleotide sequence disclosed
in SEQ ID NO: 1, more preferably in position 103-1006 of SEQ ID NO:
1.
DETAILED DESCRIPTION OF THE INVENTION
[0052] Polypeptides having OPH Activity
[0053] The present invention relates to isolated polypeptides
having organophosphatase activity comprising an amino acid sequence
which has a degree of identity to amino acids 36 to 334 of SEQ ID
NO: 2 (i.e., the mature polypeptide) of at least 87%, preferably at
least 88%, more preferably at least 89%, more preferably at least
90%, more preferably at least 91%, more preferably at least 92%,
more preferably at least 93%, more preferably at least 95%, even
more preferably at least 97%, and even most preferably at least 99%
(hereinafter "homologous polypeptides"). In a preferred aspect, the
homologous polypeptides have an amino acid sequence which differs
by ten amino acids, preferably by five amino acids, more preferably
by four amino acids, even more preferably by three amino acids,
most preferably by two amino acids, and even most preferably by one
amino acid from amino acids 36 to 334 of SEQ ID NO: 2.
[0054] A polypeptide of the present invention preferably comprises
the amino acid sequence from position 36 to 334 of SEQ ID NO: 2 or
an allelic variant thereof; or a fragment thereof that has OPH
activity. The data presented herein have identified the N-terminal
of the mature polypeptide to be either ATQQRTQ or TQQRTQV, which
shows that the mature polypeptide having OPH activity most likely
starts at position 35 in SEQ ID NO: 2, however the start could also
be at position 36 since a fragment having an N-terminal starting at
position 36 in SEQ ID NO: 2 was also observed. In a one aspect, the
polypeptide of the invention therefore consists of the amino acid
sequence of SEQ ID NO: 2. In another aspect, the polypeptide
comprises amino acids 36 to 334 of SEQ ID NO: 2, or an allelic
variant thereof; or a fragment thereof that has OPH activity. In
another preferred aspect, the polypeptide comprises amino acids 35
to 334 of SEQ ID NO: 2 or an allelic variant thereof; or a fragment
thereof that has OPH activity.
[0055] In a further aspect, the present invention relates to
isolated polypeptides having OPH activity which are encoded by
polynucleotides which hybridize under very low stringency
conditions, preferably low stringency conditions, more preferably
medium stringency conditions, more preferably medium-high
stringency conditions, even more preferably high stringency
conditions, and most preferably very high stringency conditions
with (i) nucleotides 106 to 1002 of SEQ ID NO: 1, (ii) a
subsequence of (i) of at least 100 nucleotides or (iii) a
complementary strand of (i) or (ii) (J. Sambrook, E. F. Fritsch,
and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d
edition, Cold Spring Harbor, N.Y.). A subsequence of SEQ ID NO: 1
contains at least 100 contiguous nucleotides or preferably at least
200 contiguous nucleotides. Moreover, the subsequence may encode a
polypeptide fragment which has OPH activity.
[0056] For long probes of at least 100 nucleotides in length, very
low to very high stringency conditions are defined as
prehybridization and hybridization at 42.degree. C. in
5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon
sperm DNA, and either 25% formamide for very low and low
stringencies, 35% formamide for medium and medium-high
stringencies, or 50% formamide for high and very high stringencies,
following standard Southern blotting procedures for 12 to 24 hours
optimally.
[0057] For long probes of at least 100 nucleotides in length, the
carrier material is finally washed three times each for 15 minutes
using 2.times.SSC, 0.2% SDS preferably at least at 45.degree. C.
(very low stringency), more preferably at least at 50.degree. C.
(low stringency), more preferably at least at 55.degree. C. (medium
stringency), more preferably at least at 60.degree. C. (medium-high
stringency), even more preferably at least at 65.degree. C. (high
stringency), and most preferably at least at 70.degree. C. (very
high stringency).
[0058] In a particular embodiment, the wash is conducted using
0.2.times.SSC, 0.2% SDS preferably at least at 45.degree. C. (very
low stringency), more preferably at least at 50.degree. C. (low
stringency), more preferably at least at 55.degree. C. (medium
stringency), more preferably at least at 60.degree. C. (medium-high
stringency), even more preferably at least at 65.degree. C. (high
stringency), and most preferably at least at 70.degree. C. (very
high stringency). In another particular embodiment, the wash is
conducted using 0.1.times.SSC, 0.2% SDS preferably at least at
45.degree. C. (very low stringency), more preferably at least at
50.degree. C. (low stringency), more preferably at least at
55.degree. C. (medium stringency), more preferably at least at
60.degree. C. (medium-high stringency), even more preferably at
least at 65.degree. C. (high stringency), and most preferably at
least at 70.degree. C. (very high stringency).
[0059] The nucleotide sequence of SEQ ID NO: 1 or a subsequence
thereof, as well as the amino acid sequence of SEQ ID NO: 2 or a
fragment thereof, may be used to design a nucleic acid probe to
identify and clone DNA encoding polypeptides having OPH activity
from strains of different genera or species according to methods
well known in the art. In particular, such probes can be used for
hybridization with the genomic or cDNA of the genus or species of
interest, following standard Southern blotting procedures, in order
to identify and isolate the corresponding gene therein. Such probes
can be considerably shorter than the entire sequence, but should be
at least 14, preferably at least 25, more preferably at least 35,
and most preferably at least 70 nucleotides in length. It is,
however, preferred that the nucleic acid probe is at least 100
nucleotides in length. For example, the nucleic acid probe may be
at least 200 nucleotides, preferably at least 300 nucleotides, more
preferably at least 400 nucleotides, or most preferably at least
500 nucleotides in length. Even longer probes may be used, e.g.,
nucleic acid probes which are at least 600 nucleotides, at least
preferably at least 700 nucleotides, more preferably at least 800
nucleotides, or most preferably at least 900 nucleotides in length.
Both DNA and RNA probes can be used. The probes are typically
labeled for detecting the corresponding gene (for example, with
.sup.32P, .sup.3H, .sup.35S, biotin, or avidin). Such probes are
encompassed by the present invention.
[0060] A genomic DNA or cDNA library prepared from such other
organisms may, therefore, be screened for DNA which hybridizes with
the probes described above and which encodes a polypeptide having
OPH activity. Genomic or other DNA from such other organisms may be
separated by agarose or polyacrylamide gel electrophoresis, or
other separation techniques. DNA from the libraries or the
separated DNA may be transferred to and immobilized on
nitrocellulose or other suitable carrier material. In order to
identify a clone or DNA which is homologous with SEQ ID NO: 1 or a
subsequence thereof, the carrier material is used in a Southern
blot.
[0061] For purposes of the present invention, hybridization
indicates that the nucleotide sequence hybridizes to a labeled
nucleic acid probe corresponding to the nucleotide sequence shown
in SEQ ID NO: 1, its complementary strand, or a subsequence
thereof, under very low to very high stringency conditions.
Molecules to which the nucleic acid probe hybridizes under these
conditions can be detected using X-ray film.
[0062] In a preferred aspect, the nucleic acid probe is nucleotides
106 to 1002 of SEQ ID NO: 1. In another preferred aspect, the
nucleic acid probe is a polynucleotide sequence which encodes the
polypeptide of SEQ ID NO: 2, or a subsequence thereof. In another
preferred aspect, the nucleic acid probe is SEQ ID NO: 1. In
another preferred aspect, the nucleic acid probe is the
polynucleotide sequence contained in plasmid pOPHNN10787 which is
contained in Escherichia coli DSM 17765, wherein the polynucleotide
sequence thereof encodes a polypeptide having organophosphatse
activity. In another preferred aspect, the nucleic acid probe is
the mature polypeptide coding region contained in plasmid
ppOPHNN10787.
[0063] In one aspect, the present invention relates to artificial
variants comprising a conservative substitution, deletion, and/or
insertion of one or more amino acids of SEQ ID NO: 2 or the mature
polypeptide thereof. Preferably, amino acid changes are of a minor
nature, that is conservative amino acid substitutions or insertions
that do not significantly affect the folding and/or activity of the
protein; small deletions, typically of one to about 30 amino acids;
small amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue; a small linker peptide of up to
about 20-25 residues; or a small extension that facilitates
purification by changing net charge or another function, such as a
poly-histidine tract, an antigenic epitope or a binding domain.
[0064] Examples of conservative substitutions are within the group
of basic amino acids (arginine, lysine and histidine), acidic amino
acids (glutamic acid and aspartic acid), polar amino acids
(glutamine and asparagine), hydrophobic amino acids (leucine,
isoleucine and valine), aromatic amino acids (phenylalanine,
tryptophan and tyrosine), and small amino acids (glycine, alanine,
serine, threonine and methionine).
[0065] In addition to the 20 standard amino acids, non-standard
amino acids (such as 4-hydroxyproline, 6-N-methyl lysine,
2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be
substituted for amino acid residues of a wild-type polypeptide. A
limited number of non-conservative amino acids, amino acids that
are not encoded by the genetic code, and unnatural amino acids may
be substituted for amino acid residues. "Unnatural amino acids"
have been modified after protein synthesis, and/or have a chemical
structure in their side chain(s) different from that of the
standard amino acids. Unnatural amino acids can be chemically
synthesized, and preferably, are commercially available, and
include pipecolic acid, thiazolidine carboxylic acid,
dehydroproline, 3- and 4-methylproline, and
3,3-dimethylproline.
[0066] Alternatively, the amino acid changes are of such a nature
that the physico-chemical properties of the polypeptides are
altered. For example, amino acid changes may improve the thermal
stability of the polypeptide, alter the substrate specificity,
change the pH optimum, and the like.
[0067] Essential amino acids in the parent polypeptide can be
identified according to procedures known in the art, such as
site-directed mutagenesis or alanine-scanning mutagenesis
(Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter
technique, single alanine mutations are introduced at every residue
in the molecule, and the resultant mutant molecules are tested for
biological activity (i.e., OPH activity) to identify amino acid
residues that are critical to the activity of the molecule. See
also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The
active site of the enzyme or other biological interaction can also
be determined by physical analysis of structure, as determined by
such techniques as nuclear magnetic resonance, crystallography,
electron diffraction, or photoaffinity labeling, in conjunction
with mutation of putative contact site amino acids. See, for
example, de Vos et al., 1992, Science 255: 306-312; Smith et al.,
1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett.
309:59-64. The identities of essential amino acids can also be
inferred from analysis of identities with polypeptides which are
related to a polypeptide according to the invention.
[0068] Single or multiple amino acid substitutions can be made and
tested using known methods of mutagenesis, recombination, and/or
shuffling, followed by a relevant screening procedure, such as
those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:
53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:
2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be
used include error-prone PCR, phage display (e.g., Lowman et al.,
1991, Biochem. 30:10832-10837; U.S. Pat. No. 5,223,409; WO
92/06204), and region-directed mutagenesis (Derbyshire et al.,
1986, Gene 46:145; Ner et al., 1988, DNA 7:127).
[0069] Mutagenesis/shuffling methods can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides expressed by host cells.
Mutagenized DNA molecules that encode active polypeptides can be
recovered from the host cells and rapidly sequenced using standard
methods in the art. These methods allow the rapid determination of
the importance of individual amino acid residues in a polypeptide
of interest, and can be applied to polypeptides of unknown
structure.
[0070] The total number of amino acid substitutions, deletions
and/or insertions of amino acids 36 to 334 of SEQ ID NO: 2 is 10,
preferably 9, more preferably 8, more preferably 7, more preferably
at most 6, more preferably at most 5, more preferably 4, even more
preferably 3, most preferably 2, and even most preferably 1.
Polynucleotides
[0071] The present invention also relates to isolated
polynucleotides having a nucleotide sequence which encode a
polypeptide of the present invention. In a preferred aspect, the
nucleotide sequence is set forth in SEQ ID NO: 1. In another more
preferred aspect, the nucleotide sequence is the sequence contained
in plasmid pOPHNN10787 that is contained in Escherichia coli DSM
17765. In another preferred aspect, the nucleotide sequence is the
mature polypeptide coding region of SEQ ID NO: 1. This region
should at least comprise the nucleotides from position 106 to 1002
in SEQ ID NO: 1, but could also comprise the nucleotides from
position 103 to 1002 in SEQ ID NO: 1. In another more preferred
aspect, the nucleotide sequence is the mature polypeptide coding
region contained in plasmid pOPHNN10787 that is contained in
Escherichia coli DSM 17765. The present invention also encompasses
nucleotide sequences which encode a polypeptide having the amino
acid sequence of SEQ ID NO: 2 or the mature polypeptide thereof,
which differs from SEQ ID NO: 1 by virtue of the degeneracy of the
genetic code. The present invention also relates to subsequences of
SEQ ID NO: 1 which encode fragments of SEQ ID NO: 2 that have OPH
activity.
[0072] The present invention also relates to mutant polunucleotides
comprising at least one mutation in the mature polypeptide coding
sequence of SEQ ID NO: 1, in which the mutant nucleotide sequence
encodes a polypeptide which comprises amino acids 36 to 334 of SEQ
ID NO: 2.
[0073] The techniques used to isolate or clone a polynucleotide
encoding a polypeptide are known in the art and include isolation
from genomic DNA, preparation from cDNA, or a combination thereof.
The cloning of the polynucleotides of the present invention from
such genomic DNA can be effected, e.g., by using the well known
polymerase chain reaction (PCR) or antibody screening of expression
libraries to detect cloned DNA fragments with shared structural
features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods
and Application, Academic Press, New York. Other nucleic acid
amplification procedures such as ligase chain reaction (LCR),
ligated activated transcription (LAT) and nucleotide sequence-based
amplification (NASBA) may be used. The polynucleotides may be
cloned from a strain of Ralstonia, or another or related organism
and thus, for example, may be an allelic or species variant of the
polypeptide encoding region of the nucleotide sequence.
[0074] The present invention also relates to polynucleotides having
nucleotide sequences which have a degree of identity to the mature
polypeptide coding sequence of SEQ ID NO: 1 of at least 80%, more
preferably at least 81%, more preferably at least 82%, more
preferably at least 84%, more preferably at least 86%, more
preferably at least 88%, more preferably at least 90%, more
preferably at least 92%, even more preferably at least 95%, and
most preferably at least 97% identity, which encode a polypeptide
having OPH activity. As explained herein the nucleotide sequence
encoding the mature polypeptide starts at either position 103 or
position 106 and ends at position 1002 of SEQ ID NO: 1.
[0075] Modification of a nucleotide sequence encoding a polypeptide
of the present invention may be necessary for the synthesis of
polypeptides substantially similar to the polypeptide. The term
"substantially similar" to the polypeptide refers to non-naturally
occurring forms of the polypeptide. These polypeptides may differ
in some engineered way from the polypeptide isolated from its
native source, e.g., artificial variants that differ in specific
activity, thermostability, pH optimum, or the like. The variant
sequence may be constructed on the basis of the nucleotide sequence
presented as the polypeptide encoding region of SEQ ID NO: 1, e.g.,
a subsequence thereof, and/or by introduction of nucleotide
substitutions which do not give rise to another amino acid sequence
of the polypeptide encoded by the nucleotide sequence, but which
correspond to the codon usage of the host organism intended for
production of the enzyme, or by introduction of nucleotide
substitutions which may give rise to a different amino acid
sequence. For a general description of nucleotide substitution,
see, e.g. Ford et al., 1991, Protein Expression and Purification 2:
95-107.
[0076] It will be apparent to those skilled in the art that such
substitutions can be made outside the regions critical to the
function of the molecule and still result in an active polypeptide.
Amino acid residues essential to the activity of the polypeptide
encoded by an isolated polynucleotide of the invention, and
therefore preferably not subject to substitution, may be identified
according to procedures known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham
and Wells, 1989, Science 244: 1081-1085). In the latter technique,
mutations are introduced at every positively charged residue in the
molecule, and the resultant mutant molecules are tested for OPH
activity to identify amino acid residues that are critical to the
activity of the molecule. Sites of substrate-enzyme interaction can
also be determined by analysis of the three-dimensional structure
as determined by such techniques as nuclear magnetic resonance
analysis, crystallography or photoaffinity labelling (see, e.g., de
Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal
of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS
Letters 309: 59-64).
[0077] The present invention also relates to isolated
polynucleotides encoding a polypeptide of the present invention,
which hybridize under very low stringency conditions, preferably
low stringency conditions, more preferably medium stringency
conditions, more preferably medium-high stringency conditions, even
more preferably high stringency conditions, and most preferably
very high stringency conditions with (i) nucleotides 106 to 1002 of
SEQ ID NO: 1, (ii) a subsequence of (i) of at least 100
nucleotides, or (iii) a complementary strand of (i) or (ii); and
allelic variants and subsequences thereof (Sambrook et al., 1989,
supra), as defined herein.
Nucleic Acid Constructs
[0078] The present invention also relates to nucleic acid
constructs comprising an isolated polynucleotide of the present
invention operably linked to one or more control sequences which
direct the expression of the coding sequence in a suitable host
cell under conditions compatible with the control sequences.
[0079] An isolated polynucleotide encoding a polypeptide of the
present invention may be manipulated in a variety of ways to
provide for expression of the polypeptide. Manipulation of the
polynucleotide's sequence prior to its insertion into a vector may
be desirable or necessary depending on the expression vector. The
techniques for modifying polynucleotide sequences utilizing
recombinant DNA methods are well known in the art.
[0080] The control sequence may be an appropriate promoter
sequence, a nucleotide sequence which is recognized by a host cell
for expression of a polynucleotide encoding a polypeptide of the
present invention. The promoter sequence contains transcriptional
control sequences which mediate the expression of the polypeptide.
The promoter may be any nucleotide sequence which shows
transcriptional activity in the host cell of choice including
mutant, truncated, and hybrid promoters, and may be obtained from
genes encoding extracellular or intracellular polypeptides either
homologous or heterologous to the host cell.
[0081] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention, especially in a bacterial host cell, are the promoters
obtained from the E. coli lac operon, Streptomyces coelicolor
agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),
Bacillus licheniformis alpha-amylase gene (amyL), Bacillus
stearothermophilus maltogenic amylase gene (amyM), Bacillus
amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis
penicillinase gene (penP), Bacillus subtilis xylA and xylB genes,
and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,
Proceedings of the National Academy of Sciences USA 75: 3727-3731),
as well as the tac promoter (DeBoer et al., 1983, Proceedings of
the National Academy of Sciences USA 80: 21-25). Further promoters
are described in "Useful proteins from recombinant bacteria" in
Scientific American, 1980, 242: 74-94; and in Sambrook et al.,
1989, supra.
[0082] Examples of suitable promoters for directing the
transcription of the nucleic acid constructs of the present
invention in a filamentous fungal host cell are promoters obtained
from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900),
Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn
(WO 00/56900), Fusarium oxysporum trypsin-like protease (WO
96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei
cellobiohydrolase I, Trichoderma reesei endoglucanase I,
Trichoderma reesei endoglucanase II, Trichoderma reesei
endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma
reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma
reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as
the NA2-tpi promoter (a hybrid of the promoters from the genes for
Aspergillus niger neutral alpha-amylase and Aspergillus oryzae
triose phosphate isomerase); and mutant, truncated, and hybrid
promoters thereof.
[0083] In a yeast host, useful promoters are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,
ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase
(TPI), Saccharomyces cerevisiae metallothionine (CUP1), and
Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful
promoters for yeast host cells are described by Romanos et al.,
1992, Yeast 8: 423-488.
[0084] The control sequence may also be a suitable transcription
terminator sequence, a sequence recognized by a host cell to
terminate transcription. The terminator sequence is operably linked
to the 3' terminus of the nucleotide sequence encoding the
polypeptide. Any terminator which is functional in the host cell of
choice may be used in the present invention.
[0085] Preferred terminators for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase,
Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate
synthase, Aspergillus niger alpha-glucosidase, and Fusarium
oxysporum trypsin-like protease.
[0086] Preferred terminators for yeast host cells are obtained from
the genes for Saccharomyces cerevisiae enolase, Saccharomyces
cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae
glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators
for yeast host cells are described by Romanos et al., 1992,
supra.
[0087] The control sequence may also be a suitable leader sequence,
a nontranslated region of an mRNA which is important for
translation by the host cell. The leader sequence is operably
linked to the 5' terminus of the nucleotide sequence encoding the
polypeptide. Any leader sequence that is functional in the host
cell of choice may be used in the present invention.
[0088] Preferred leaders for filamentous fungal host cells are
obtained from the genes for Aspergillus oryzae TAKA amylase and
Aspergillus nidulans triose phosphate isomerase.
[0089] Suitable leaders for yeast host cells are obtained from the
genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces
cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae
alpha-factor, and Saccharomyces cerevisiae alcohol
dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
(ADH2/GAP).
[0090] The control sequence may also be a polyadenylation sequence,
a sequence operably linked to the 3' terminus of the nucleotide
sequence and which, when transcribed, is recognized by the host
cell as a signal to add polyadenosine residues to transcribed mRNA.
Any polyadenylation sequence which is functional in the host cell
of choice may be used in the present invention.
[0091] Preferred polyadenylation sequences for filamentous fungal
host cells are obtained from the genes for Aspergillus oryzae TAKA
amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Fusarium oxysporum trypsin-like protease,
and Aspergillus niger alpha-glucosidase.
[0092] Useful polyadenylation sequences for yeast host cells are
described by Guo and Sherman, 1995, Molecular Cellular Biology 15:
5983-5990.
[0093] The control sequence may also be a signal peptide coding
region that codes for an amino acid sequence linked to the amino
terminus of a polypeptide and directs the encoded polypeptide into
the cell's secretory pathway. The 5' end of the coding sequence of
the nucleotide sequence may inherently contain a signal peptide
coding region naturally linked in translation reading frame with
the segment of the coding region which encodes the secreted
polypeptide. Alternatively, the 5' end of the coding sequence may
contain a signal peptide coding region which is foreign to the
coding sequence. The foreign signal peptide coding region may be
required where the coding sequence does not naturally contain a
signal peptide coding region. Alternatively, the foreign signal
peptide coding region may simply replace the natural signal peptide
coding region in order to enhance secretion of the polypeptide.
However, any signal peptide coding region which directs the
expressed polypeptide into the secretory pathway of a host cell of
choice may be used in the present invention.
[0094] Effective signal peptide coding regions for bacterial host
cells are the signal peptide coding regions obtained from the genes
for Bacillus NCIB 11837 maltogenic amylase, Bacillus
stearothermophilus alpha-amylase, Bacillus licheniformis
subtilisin, Bacillus licheniformis beta-lactamase, Bacillus
stearothermophilus neutral proteases (nprT, nprS, nprM), and
Bacillus subtilis prsA. Further signal peptides are described by
Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
[0095] Effective signal peptide coding regions for filamentous
fungal host cells are the signal peptide coding regions obtained
from the genes for Aspergillus oryzae TAKA amylase, Aspergillus
niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor
miehei aspartic proteinase, Humicola insolens cellulase, and
Humicola lanuginosa lipase.
[0096] The OPH of the present invention as shown in SEQ ID NO 2 and
encoded for by SEQ ID NO 1 comprises its natural signal peptide
coding region. This native signal peptide consists of amino acids 1
to 34 and maybe even 35 of SEQ ID NO 2, encoded for by nucleotides
1 to 102 or 105 in SEQ ID NO 1. Since the origin of the OPH
according to the invention is bacterial the native signal peptide
is particularly useful for expression of the OPH in a bacterial
host cell. However, as evidenced by the data presented in the
examples, this native bacterial signal peptide will also result in
secretion of the OPH when expressed in a fungal host cell.
[0097] Useful signal peptides for yeast host cells are obtained
from the genes for Saccharomyces cerevisiae alpha-factor and
Saccharomyces cerevisiae invertase. Other useful signal peptide
coding regions are described by Romanos et al., 1992, supra.
[0098] The control sequence may also be a propeptide coding region
that codes for an amino acid sequence positioned at the amino
terminus of a polypeptide. The resultant polypeptide is known as a
proenzyme or propolypeptide (or a zymogen in some cases). A
propolypeptide is generally inactive and can be converted to a
mature active polypeptide by catalytic or autocatalytic cleavage of
the propeptide from the propolypeptide. The propeptide coding
region may be obtained from the genes for Bacillus subtilis
alkaline protease (aprE), Bacillus subtilis neutral protease
(nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei
aspartic proteinase, and Myceliophthora thermophila laccase (WO
95/33836).
[0099] Where both signal peptide and propeptide regions are present
at the amino terminus of a polypeptide, the propeptide region is
positioned next to the amino terminus of a polypeptide and the
signal peptide region is positioned next to the amino terminus of
the propeptide region.
[0100] It may also be desirable to add regulatory sequences which
allow the regulation of the expression of the polypeptide relative
to the growth of the host cell. Examples of regulatory systems are
those which cause the expression of the gene to be turned on or off
in response to a chemical or physical stimulus, including the
presence of a regulatory compound. Regulatory systems in
prokaryotic systems include the lac, tac, and trp operator systems.
In yeast, the ADH2 system or GAL1 system may be used. In
filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus
niger glucoamylase promoter, and Aspergillus oryzae glucoamylase
promoter may be used as regulatory sequences. Other examples of
regulatory sequences are those which allow for gene amplification.
In eukaryotic systems, these include the dihydrofolate reductase
gene which is amplified in the presence of methotrexate, and the
metallothionein genes which are amplified with heavy metals. In
these cases, the nucleotide sequence encoding the polypeptide would
be operably linked with the regulatory sequence.
Expression Vectors
[0101] The present invention also relates to recombinant expression
vectors comprising a polynucleotide of the present invention, a
promoter, and transcriptional and translational stop signals. The
various nucleic acids and control sequences described above may be
joined together to produce a recombinant expression vector which
may include one or more convenient restriction sites to allow for
insertion or substitution of the nucleotide sequence encoding the
polypeptide at such sites. Alternatively, a nucleotide sequence of
the present invention may be expressed by inserting the nucleotide
sequence or a nucleic acid construct comprising the sequence into
an appropriate vector for expression. In creating the expression
vector, the coding sequence is located in the vector so that the
coding sequence is operably linked with the appropriate control
sequences for expression.
[0102] The recombinant expression vector may be any vector (e.g., a
plasmid or virus) which can be conveniently subjected to
recombinant DNA procedures and can bring about expression of the
nucleotide sequence. The choice of the vector will typically depend
on the compatibility of the vector with the host cell into which
the vector is to be introduced. The vectors may be linear or closed
circular plasmids.
[0103] The vector may be an autonomously replicating vector, i.e.,
a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids which together contain the total DNA to
be introduced into the genome of the host cell, or a transposon may
be used.
[0104] The vectors of the present invention preferably contain one
or more selectable markers which permit easy selection of
transformed cells. A selectable marker is a gene the product of
which provides for biocide or viral resistance, resistance to heavy
metals, prototrophy to auxotrophs, and the like.
[0105] A conditionally essential gene may function as a
non-antibiotic selectable marker. Non-limiting examples of
bacterial conditionally essential non-antibiotic selectable markers
are the dal genes from Bacillus subtilis, Bacillus licheniformis,
or other Bacilli, that are only essential when the bacterium is
cultivated in the absence of D-alanine. Also the genes encoding
enzymes involved in the turnover of UDP-galactose can function as
conditionally essential markers in a cell when the cell is grown in
the presence of galactose or grown in a medium which gives rise to
the presence of galactose. Non-limiting examples of such genes are
those from B. subtilis or B. licheniformis encoding UTP-dependent
phosphorylase (EC 2.7.7.10), UDP-glucose-dependent
uridylyltransferase (EC 2.7.7.12), or UDP-galactose epimerase (EC
5.1.3.2). Also a xylose isomerase gene such as xylA, of Bacilli can
be used as selectable markers in cells grown in minimal medium with
xylose as sole carbon source. The genes necessary for utilizing
gluconate, gntK, and gntP can also be used as selectable markers in
cells grown in minimal medium with gluconate as sole carbon source.
Other examples of conditionally essential genes are known in the
art. Antibiotic selectable markers confer antibiotic resistance to
such antibiotics as ampicillin, kanamycin, chloramphenicol,
erythromycin, tetracycline, neomycin, hygromycin or
methotrexate.
[0106] Suitable markers for yeast host cells are ADE2, HIS3, LEU2,
LYS2, MET3, TRP1, and URA3. Selectable markers for use in a
filamentous fungal host cell include, but are not limited to, amdS
(acetamidase), argB (ornithine carbamoyltransferase), bar
(phosphinothricin acetyltransferase), hph (hygromycin
phosphotransferase), niaD (nitrate reductase), pyrG
(orotidine-5'-phosphate decarboxylase), sC (sulfate
adenyltransferase), and trpC (anthranilate synthase), as well as
equivalents thereof. Preferred for use in an Aspergillus cell are
the amdS and pyrG genes of Aspergillus nidulans or Aspergillus
oryzae and the bar gene of Streptomyces hygroscopicus.
[0107] The vectors of the present invention preferably contain an
element(s) that permits integration of the vector into the host
cell's genome or autonomous replication of the vector in the cell
independent of the genome.
[0108] For integration into the host cell genome, the vector may
rely on the polynucleotide's sequence encoding the polypeptide or
any other element of the vector for integration into the genome by
homologous or nonhomologous recombination. Alternatively, the
vector may contain additional nucleotide sequences for directing
integration by homologous recombination into the genome of the host
cell at a precise location(s) in the chromosome(s). To increase the
likelihood of integration at a precise location, the integrational
elements should preferably contain a sufficient number of nucleic
acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000
base pairs, and most preferably 800 to 10,000 base pairs, which
have a high degree of identity with the corresponding target
sequence to enhance the probability of homologous recombination.
The integrational elements may be any sequence that is homologous
with the target sequence in the genome of the host cell.
Furthermore, the integrational elements may be non-encoding or
encoding nucleotide sequences. On the other hand, the vector may be
integrated into the genome of the host cell by non-homologous
recombination.
[0109] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. The origin of
replication may be any plasmid replicator mediating autonomous
replication which functions in a cell. The term "origin of
replication" or "plasmid replicator" is defined herein as a
nucleotide sequence that enables a plasmid or vector to replicate
in vivo.
[0110] Examples of bacterial origins of replication are the origins
of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184
permitting replication in E. coli, and pUB110, pE194, pTA1060, and
pAM.beta.1 permitting replication in Bacillus.
[0111] Examples of origins of replication for use in a yeast host
cell are the 2 micron origin of replication, ARS1, ARS4, the
combination of ARS1 and CEN3, and the combination of ARS4 and
CEN6.
[0112] Examples of origins of replication useful in a filamentous
fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67;
Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO
00/24883). Isolation of the AMA1 gene and construction of plasmids
or vectors comprising the gene can be accomplished according to the
methods disclosed in WO 00/24883.
[0113] More than one copy of a polynucleotide of the present
invention may be inserted into the host cell to increase production
of the gene product. An increase in the copy number of the
polynucleotide can be obtained by integrating at least one
additional copy of the sequence into the host cell genome or by
including an amplifiable selectable marker gene with the
polynucleotide where cells containing amplified copies of the
selectable marker gene, and thereby additional copies of the
polynucleotide, can be selected for by cultivating the cells in the
presence of the appropriate selectable agent.
[0114] The procedures used to ligate the elements described above
to construct the recombinant expression vectors of the present
invention are well known to one skilled in the art (see, e.g.,
Sambrook et al., 1989, supra).
Host Cells
[0115] The present invention also relates to recombinant host
cells, comprising a polynucleotide of the present invention, which
are advantageously used in the recombinant production of the
polypeptides. A vector comprising a polynucleotide of the present
invention is introduced into a host cell so that the vector is
maintained as a chromosomal integrant or as a self-replicating
extra-chromosomal vector as described earlier. The term "host cell"
encompasses any progeny of a parent cell that is not identical to
the parent cell due to mutations that occur during replication. The
choice of a host cell will to a large extent depend upon the gene
encoding the polypeptide and its source.
[0116] The host cell may be a prokaryote or a eukaryote. The host
cell may be unicellular.
[0117] Useful unicellular microorganisms are bacterial cells such
as gram positive bacteria including, but not limited to, a Bacillus
cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens,
Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus
coagulans, Bacillus lautus, Bacillus lentus, Bacillus
licheniformis, Bacillus megaterium, Bacillus stearothermophilus,
Bacillus subtilis, and Bacillus thuringiensis; or a Streptomyces
cell, e.g., Streptomyces lividans and Streptomyces murinus, or gram
negative bacteria such as E. coli and Pseudomonas sp. In a
preferred aspect, the bacterial host cell is a Bacillus lentus,
Bacillus licheniformis, Bacillus stearothermophilus, or Bacillus
subtilis cell. In another preferred aspect, the Bacillus cell is an
alkalophilic Bacillus.
[0118] The introduction of a vector into a bacterial host cell may,
for instance, be effected by protoplast transformation (see, e.g.,
Chang and Cohen, 1979, Molecular General Genetics 168: 111-115),
using competent cells (see, e.g., Young and Spizizin, 1961, Journal
of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971,
Journal of Molecular Biology 56: 209-221), electroporation (see,
e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or
conjugation (see, e.g., Koehler and Thorne, 1987, Journal of
Bacteriology 169: 5771-5278).
[0119] The host cell may also be a eukaryote, such as a mammalian,
insect, plant, or fungal cell.
[0120] In a preferred aspect, the host cell is a fungal cell.
"Fungi" as used herein includes the phyla Ascomycota,
Basidiomycota, Chytridiomycota, and Zygomycota (as defined by
Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The
Fungi, 8th edition, 1995, CAB International, University Press,
Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et
al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et
al., 1995, supra).
[0121] In a more preferred aspect, the fungal host cell is a yeast
cell. "Yeast" as used herein includes ascosporogenous yeast
(Endomycetales), basidiosporogenous yeast, and yeast belonging to
the Fungi Imperfecti (Blastomycetes). Since the classification of
yeast may change in the future, for the purposes of this invention,
yeast shall be defined as described in Biology and Activities of
Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds,
Soc. App. Bacteriol. Symposium Series No. 9, 1980).
[0122] In an even more preferred aspect, the yeast host cell is a
Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,
Schizosaccharomyces, or Yarrowia cell.
[0123] In a most preferred aspect, the yeast host cell is a
Saccharomyces carlsbergensis, Saccharomyces cerevisiae,
Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces
kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell.
In another most preferred aspect, the yeast host cell is a
Kluyveromyces lactis cell. In another most preferred aspect, the
yeast host cell is a Yarrowia lipolytica cell.
[0124] In another more preferred aspect, the fungal host cell is a
filamentous fungal cell. "Filamentous fungi" include all
filamentous forms of the subdivision Eumycota and Oomycota (as
defined by Hawksworth et al., 1995, supra). The filamentous fungi
are generally characterized by a mycelial wall composed of chitin,
cellulose, glucan, chitosan, mannan, and other complex
polysaccharides. Vegetative growth is by hyphal elongation and
carbon catabolism is obligately aerobic. In contrast, vegetative
growth by yeasts such as Saccharomyces cerevisiae is by budding of
a unicellular thallus and carbon catabolism may be
fermentative.
[0125] In an even more preferred aspect, the filamentous fungal
host cell is an Acremonium, Aspergillus, Aureobasidium,
Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus,
Filobasidium, Fusarium, Humicola, Magnaporthe, Mucor,
Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,
Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,
Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,
Trametes, or Trichoderma cell.
[0126] In a most preferred aspect, the filamentous fungal host cell
is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus
foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus
niger or Aspergillus oryzae cell. In another most preferred aspect,
the filamentous fungal host cell is a Fusarium bactridioides,
Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum,
Fusarium graminearum, Fusarium graminum, Fusarium heterosporum,
Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum,
Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,
Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,
Fusarium trichothecioldes, or Fusarium venenatum cell. In another
most preferred aspect, the filamentous fungal host cell is a
Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina,
Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis
pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, or
Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,
Humicola insolens, Humicola lanuginosa, Mucor miehei,
Myceliophthora thermophila, Neurospora crassa, Penicillium
purpurogenum, Phanerochaete chrysosporium, Phlebia radiata,
Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes
versicolor, Trichoderma harzianum, Trichoderma koningii,
Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma
viride cell.
[0127] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts, and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus and Trichoderma host
cells are described in EP 238 023 and Yelton et al., 1984,
Proceedings of the National Academy of Sciences USA 81: 1470-1474.
Suitable methods for transforming Fusarium species are described by
Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast
may be transformed using the procedures described by Becker and
Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to
Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume
194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983,
Journal of Bacteriology 153: 163; and Hinnen et al., 1978,
Proceedings of the National Academy of Sciences USA 75: 1920.
Plants
[0128] The present invention also relates to a transgenic plant,
plant part, or plant cell which has been transformed with a
nucleotide sequence encoding a polypeptide having OPH activity of
the present invention so as to express and produce the polypeptide
in recoverable quantities. The polypeptide may be recovered from
the plant or plant part.
[0129] Plant organisms for the purposes of the invention are
organisms capable of being photosynthetically active such as, for
example, algae, cyanobacteria and mosses. Preferred algae are green
algae such as, for example, algae from the genus Haematococcus,
Phaedactylum tricornatum, Volvox or Dunaliella.
[0130] The transgenic plant can be dicotyledonous (a dicot) or
monocotyledonous (a monocot). Examples of monocot plants are
grasses, such as meadow grass (blue grass, Poa), forage grass such
as Festuca, Lolium, temperate grass, such as Agrostis, and cereals,
e.g., wheat, oats, rye, barley, rice, sorghum, and maize
(corn).
[0131] Examples of dicot plants are tobacco, legumes, such as
lupins, potato, sugar beet, pea, bean and soybean, and cruciferous
plants (family Brassicaceae), such as cauliflower, rape seed, and
the closely related model organism Arabidopsis thaliana.
[0132] Examples of plant parts are stem, callus, leaves, root,
fruits, seeds, and tubers as well as the individual tissues
comprising these parts, e.g., epidermis, mesophyll, parenchyme,
vascular tissues, meristems. Specific plant cell compartments, such
as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and
cytoplasm are also considered to be a plant part. Furthermore, any
plant cell, whatever the tissue origin, is considered to be a plant
part. Likewise, plant parts such as specific tissues and cells
isolated to facilitate the utilisation of the invention are also
considered plant parts, e.g., embryos, endosperms, aleurone and
seeds coats.
[0133] Also included within the scope of the present invention are
the progeny of such plants, plant parts, and plant cells.
[0134] The transgenic plant or plant cell expressing a polypeptide
of the present invention may be constructed in accordance with
methods known in the art. In short, the plant or plant cell is
constructed by incorporating one or more expression constructs
encoding a polypeptide of the present invention into the plant host
genome and propagating the resulting modified plant or plant cell
into a transgenic plant or plant cell.
[0135] The expression construct is conveniently a nucleic acid
construct which comprises a polynucleotide encoding a polypeptide
of the present invention operably linked with appropriate
regulatory sequences required for expression of the nucleotide
sequence in the plant or plant part of choice. Furthermore, the
expression construct may comprise a selectable marker useful for
identifying host cells into which the expression construct has been
integrated and DNA sequences necessary for introduction of the
construct into the plant in question (the latter depends on the DNA
introduction method to be used).
[0136] Cloning vectors and techniques for the genetic manipulation
of ciliates and algae are known to the skilled worker (WO 98/01572;
Falciatore et al. (1999) Marine Biotechnology 1 (3): 239-251;
Dunahay et al. (1995) J Phycol 31: 10004-1012; Mayfield et al.,
2003, PNAS 100 (2): 438-442).
[0137] The choice of regulatory sequences, such as promoter and
terminator sequences and optionally signal or transit sequences are
determined, for example, on the basis of when, where, and how the
polypeptide is desired to be expressed. For instance, the
expression of the gene encoding a polypeptide of the present
invention may be constitutive or inducible, or may be
developmental, stage or tissue specific, and the gene product may
be targeted to a specific tissue or plant part such as seeds or
leaves. Regulatory sequences are, for example, described by Tague
et al., 1988, Plant Physiology 86: 506.
[0138] For constitutive expression, the 35S-CaMV, the maize
ubiquitin 1, and the rice actin 1 promoter may be used (Franck et
al., 1980, Cell 21: 285-294, Christensen et al., 1992, Plant Mo.
Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165).
Organ-specific promoters may be, for example, a promoter from
storage sink tissues such as seeds, potato tubers, and fruits
(Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or
from metabolic sink tissues such as meristems (Ito et al., 1994,
Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the
glutelin, prolamin, globulin, or albumin promoter from rice (Wu et
al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba
promoter from the legumin B4 and the unknown seed protein gene from
Vicia faba (Conrad et al., 1998, Journal of Plant Physiology 152:
708-711), a promoter from a seed oil body protein (Chen et al.,
1998, Plant and Cell Physiology 39: 935-941), the storage protein
napA promoter from Brassica napus, or any other seed specific
promoter known in the art, e.g., as described in WO 91/14772.
Furthermore, the promoter may be a leaf specific promoter such as
the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant
Physiology 102: 991-1000, the chlorella virus adenine
methyltransferase gene promoter (Mitra and Higgins, 1994, Plant
Molecular Biology 26: 85-93), or the aldP gene promoter from rice
(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674),
or a wound inducible promoter such as the potato pin2 promoter (Xu
et al., 1993, Plant Molecular Biology 22: 573-588). Likewise, the
promoter may inducible by abiotic treatments such as temperature,
drought, or alterations in salinity or induced by exogenously
applied substances that activate the promoter, e.g., ethanol,
oestrogens, plant hormones such as ethylene, abscisic acid, and
gibberellic acid, and heavy metals.
[0139] A promoter enhancer element may also be used to achieve
higher expression of a polypeptide of the present invention in the
plant. For instance, the promoter enhancer element may be an intron
which is placed between the promoter and the nucleotide sequence
encoding a polypeptide of the present invention. For instance, Xu
et al., 1993, supra, disclose the use of the first intron of the
rice actin 1 gene to enhance expression.
[0140] The selectable marker gene and any other parts of the
expression construct may be chosen from those available in the
art.
[0141] The nucleic acid construct is incorporated into the plant
genome according to conventional techniques known in the art,
including Agrobacterium-mediated transformation, virus-mediated
transformation, microinjection, particle bombardment, biolistic
transformation, and electroporation (Gasser et al., 1990, Science
244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al.,
1989, Nature 338: 274).
[0142] Presently, Agrobacterium tumefaciens-mediated gene transfer
is the method of choice for generating transgenic dicots (for a
review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology
19: 15-38) and can also be used for transforming monocots, although
other transformation methods are often used for these plants.
Presently, the method of choice for generating transgenic monocots
is particle bombardment (microscopic gold or tungsten particles
coated with the transforming DNA) of embryonic calli or developing
embryos (Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994,
Current Opinion Biotechnology 5: 158-162; Vasil et al., 1992,
Bio/Technology 10: 667-674). An alternative method for
transformation of monocots is based on protoplast transformation as
described by Omirulleh etal., 1993, Plant MolecularBiology 21:
415-428.
[0143] Following transformation, the transformants having
incorporated the expression construct are selected and regenerated
into whole plants according to methods well-known in the art. Often
the transformation procedure is designed for the selective
elimination of selection genes either during regeneration or in the
following generations by using, for example, co-transformation with
two separate T-DNA constructs or site specific excision of the
selection gene by a specific recombinase.
[0144] The present invention also relates to methods for producing
a polypeptide of the present invention comprising (a) cultivating a
transgenic plant or a plant cell comprising a polynucleotide
encoding a polypeptide having organophosphatase activity of the
present invention under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
[0145] In a further embodiment the present invention relates to a
method for producing the polypeptide of the invention, comprising
(a) cultivating an algae comprising a polynucleotide encoding a
polypeptide having organophosphatase activity of the present
invention under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide.
Methods of Production
[0146] The present invention also relates to methods for producing
a polypeptide of the present invention, comprising (a) cultivating
a cell, which in its wild-type form is capable of producing the
polypeptide, under conditions conducive for production of the
polypeptide; and (b) recovering the polypeptide. Preferably, the
cell is of the genus Ralstonia, and more preferably Ralstonia
pickettii.
[0147] The present invention also relates to methods for producing
a polypeptide of the present invention, comprising (a) cultivating
a host cell comprising a nucleic acid construct comprising a
nucleotide sequence encoding the polypeptide of the invention under
conditions conducive for production of the polypeptide; and (b)
recovering the polypeptide.
[0148] The present invention also relates to methods for producing
a polypeptide of the present invention, comprising (a) cultivating
a host cell under conditions conducive for production of the
polypeptide, wherein the host cell comprises a mutant nucleotide
sequence having at least one mutation in the mature polypeptide
coding region of SEQ ID NO: 1, wherein the mutant nucleotide
sequence encodes a polypeptide which comprises amino acids 36 to
334 of SEQ ID NO: 2, and (b) recovering the polypeptide.
[0149] In the production methods of the present invention, the
cells are cultivated in a nutrient medium suitable for production
of the polypeptide using methods well known in the art. For
example, the cell may be cultivated by shake flask cultivation, and
small-scale or large-scale fermentation (including continuous,
batch, fed-batch, or solid state fermentations) in laboratory or
industrial fermentors performed in a suitable medium and under
conditions allowing the polypeptide to be expressed and/or
isolated. The cultivation takes place in a suitable nutrient medium
comprising carbon and nitrogen sources and inorganic salts, using
procedures known in the art. Suitable media are available from
commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection). If the polypeptide is secreted into the nutrient
medium, the polypeptide can be recovered directly from the medium.
If the polypeptide is not secreted, it can be recovered from cell
lysates.
[0150] The polypeptides may be detected using methods known in the
art that are specific for the polypeptides. These detection methods
may include use of specific antibodies, formation of an enzyme
product, or disappearance of an enzyme substrate. For example, an
enzyme assay may be used to determine the activity of the
polypeptide as described herein.
[0151] The resulting polypeptide may be recovered using methods
known in the art. For example, the polypeptide may be recovered
from the nutrient medium by conventional procedures including, but
not limited to, centrifugation, filtration, extraction,
spray-drying, evaporation, or precipitation.
[0152] The polypeptides of the present invention may be purified by
a variety of procedures known in the art including, but not limited
to, chromatography (e.g., ion exchange, affinity, hydrophobic,
chromatofocusing, and size exclusion), electrophoretic procedures
(e.g., preparative isoelectric focusing), differential solubility
(e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction
(see, e.g., Protein Purification, J.-C. Janson and Lars Ryden,
editors, VCH Publishers, New York, 1989).
Compositions
[0153] The present invention also relates to compositions
comprising a polypeptide of the present invention. Preferably, the
compositions are enriched in such a polypeptide. The term
"enriched" indicates that the OPH activity of the composition has
been increased, e.g. with an enrichment factor of 1.1.
[0154] The composition may comprise a polypeptide of the present
invention as the major enzymatic component, e.g., a mono-component
composition. Alternatively, the composition may comprise multiple
enzymatic activities, such as an aminopeptidase, amylase,
carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,
cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,
esterase, alpha-galactosidase, beta-galactosidase, glucoamylase,
alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase,
laccase, lipase, mannosidase, oxidase, pectinolytic enzyme,
peptidoglutaminase, peroxidase, phytase, polyphenoloxidase,
proteolytic enzyme, ribonuclease, transglutaminase, urease, or
xylanase. The additional enzyme(s) may be produced, for example, by
a microorganism belonging to the genus Aspergillus, preferably
Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,
Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,
Aspergillus niger, or Aspergillus oryzae; Fusarium, preferably
Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,
Fusarium culmorum, Fusarium graminearum, Fusarium graminum,
Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum,
Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,
Fusarium sarcochroum, Fusarium sulphureum, Fusarium toruloseum,
Fusarium trichothecioides, or Fusarium venenatum; Humicola,
preferably Humicola insolens or Humicola lanuginosa; or
Trichoderma, preferably Trichoderma harzianum, Trichoderma
koningii, Trichoderma longibrachiatum, Trichoderma reesei, or
Trichoderma viride.
[0155] In a particular embodiment the invention relates to a
composition for hydrolysing an organophosphatase molecule, said
composition comprising a polypeptide according to the invention and
one or more acceptable carriers.
[0156] In another particular embodiment the composition comprises a
host cell expressing the organophosphatase of the invention.
[0157] The polypeptide compositions may be prepared in accordance
with methods known in the art and may be in the form of a liquid or
a dry composition. For instance, the polypeptide composition may be
in the form of a granulate or a microgranulate. The polypeptide to
be included in the composition may be stabilized in accordance with
methods known in the art.
[0158] Examples are given below of preferred uses of the
polypeptide compositions of the invention. The dosage of the
polypeptide composition of the invention and other conditions under
which the composition is used may be determined on the basis of
methods known in the art.
Uses
[0159] The present invention is also directed to methods for using
the polypeptides having organophosphatase activity for hydrolysing
an organophosphorus molecule by contacting the said molecule with
the organophosphatase.
[0160] Exposing or contacting the the organophosphorus molecule to
the organophasphatase according to the invention can be performed
in many ways, e.g. by exposing a composition or a host cell
comprising the OPH to the said molecule.
[0161] The present invention is further described by the following
examples which should not be construed as limiting the scope of the
invention.
EXAMPLES
[0162] Chemicals used as buffers and substrates were commercial
products of at least reagent grade.
Example 1
Cloning and Expression of OPH from Ralstonia picketii ATCC27511
TABLE-US-00002 [0163] Reagents and media Nutrient agar Peptone 5.0
g Meat extract 3.0 g Agar 15.0 g Distilled water 1000.0 ml Adjust
pH to 7.0. Autoclave at 121 .degree. C., 16 minutes Nutrient broth:
Peptone 5.0 g Meat extract 3.0 g Distilled water 1000.0 ml Adjust
pH to 7.0. Autoclave at 121.degree. C., 16 minutes TE 10 mM
Tris-HCl, pH 7.4 1 mM EDTA, pH 8.0 TEL 50 mg/ml Lysozym in
TE-buffer Thiocyanate 5M guanidium thiocyanate 100 mM EDTA 0.6% w/v
N-laurylsarcosine, sodium salt 60 g thiocyanate, 20 ml 0.5 M EDTA,
pH 8.0, 20 ml H.sub.2O dissolves at 65.degree. C. Cool down to room
temperature (RT) and add 0.6 g N-laurylsarcosine. Add H.sub.2O to
100 ml and filter it through a 0.2 .mu. sterile filter. NH.sub.4Ac
7.5 M CH.sub.3COONH.sub.4 TER 1 .mu.g/ml RNAse A in TE-buffer CIA
Chloroform/isoamyl alcohol 24:1 TY*2 medium Tryptone 40 g Yeast
Extract 10 g 1% Ferrochloride 1.4 ml 1% Mangan(II)-chloride 0.2 ml
1% Magnesiumsulfate 3.0 ml Add distilled water up to 1000 ml;
adjust pH to 7.3 and autoclave at 121.degree. C., 16 minutes.
Lysisbuffer: HEPES 100 mM pH 7 200 .mu.l Triton X-100 20 .mu.l
DNA{acute over ( )}se 10 mg/ml 2 .mu.l Lysozym 10 mg/ml 2 .mu.l Add
distilled water up to 1 ml DNA{acute over ( )}se 10 mg/ml in 50%
glycerol an 10 mM Hepes pH 7.
Cloning of SEQ ID NO: 1
[0164] SEQ ID NO: 1 is the DNA sequence encoding the OPH from
Ralstonia picketii ATCC27511. Genomic DNA from Ralstonia picketii
can be isolated according to the following procedure from an over
night culture in nutrient broth at 37.degree. C.: [0165] 1. Harvest
1.5 ml culture and resuspend in 100 .mu.l TEL. Incubate at
37.degree. C. for 30 min. [0166] 2. Add 500 .mu.l thiocynate buffer
and leave at room temperature for 10 min. [0167] 3. Add 250 .mu.l
NH.sub.4Ac and leave at ice for 10 min. [0168] 4. Add 500 .mu.l CIA
and mix. [0169] 5. Transfer to a microcentrifuge and spin for 10
min. at full speed. [0170] 6. Transfer supernatant to a new
Eppendorf tube and add 0.54 volume cold isopropanol. Mix
thoroughly. [0171] 7. Spin and wash the DNA pellet with 70% EtOH.
[0172] 8. Resuspend the genomic DNA in 100 .mu.l TER. The genomic
DNA from Ralstonia picketii ATCC27511 can be used as template for
PCR amplification of the OPH Ralstonia picketii ATCC27511 gene by
standard PCR methods using primer 1918 and primer 1919.
Primer 1918 (SEQ ID NO 3):
[0172] [0173] 5'-GGATTAATGCGTTTGCTTACTTCTGTCGCCGTG -3', start codon
in bold and restriction site Ase I is underlined
Primer 1919 (SEQ ID NO 4):
[0173] [0174] 5'-CCGCTCGAGATCAGCGCGGTCTGTGCGCAG-3', restriction
site Xho I is underlined. Please note that using primer 1919 the
stop codon TGA is removed, useful for expression with C-terminal
His-tag's. The PCR product amplified with primer 1918 and 1919 was
digested with restriction enzyme Ase I and Xho I using standard
method, and the resulting fragment was ligated to Nde I/Xho I
digested pET-30a(+) (Novagen) vector prior to transformation into
E. coli DH10B. E. coli DH10B Kanamycin resistant transformants were
further analyzed by DNA sequencing of the OPH gene insert in the
pET-30a(+). A plasmid with correct sequence, pOPHNN10787, was
selected.
Fermentation
[0175] pOPHNN10787 was transformed into E. coli BL21 according to
standard transformation protocols. A correct transformant, E. coli
BL21 (pOPHNN10787), was selected by ampicillin selection.
Recombinant expression of the His tag OPH from Ralstonia picketii
ATCC27511 was done from E. coli BL21 (pOPHNN10787) according to
standard protocols (see e.g. the pET System Manual).
Lysis of Cells
[0176] Cells were harvested at 4000 rpm, 30 min. Cell pellet was
resuspended in lysis buffer (a 1/10 of the culture volume) and
transferred to 37.degree. C. under shaking (250 rpm) for 15
minutes. The samples were centrifuged (4000 rpm, 10 minutes) and
the supernatants were collected. The level of expression was
analysed by standard SDS PAGE analysis (no visible band) and the
supernatants were used for the paraoxonase assay as described in
Example 2.
Example 2
Expression and Purification of OPH from Aspergillus oryzae
TABLE-US-00003 [0177] Reagent and media Trace element mixture (100
.times. concentrate) MgCl.sub.2--6H.sub.2O 12.5 g CaCl2 0.55 g
FeCl.sub.3--6H.sub.2O 1.35 g MnCl.sub.2--4H.sub.2O 0.1 g ZnCl.sub.2
0.17 g CuCl.sub.2--2H.sub.2O 0.043 g CoCl.sub.2--6H.sub.2O 0.06 g
Na.sub.2MoO.sub.4--2H.sub.2O 0.06 g YP medium Yeast extract 10 g
Peptone 20 g Ion exchanged water to 1 L Make sure pH is 6.8 as
expected, then autoclave medium
Construction of Plasmid
[0178] Plasmid DNA of pOPHNN10787 can be prepared by standard
methods. The obtained plasmid can then be used as template in a
standard PCR reaction using either primer 106 and 107 or primer 121
and 107.
Primer 106 (SEQ ID NO 5):
[0179] 5'-GTA GGA TCC ACC ATG CGT TTG CTT ACT TCT GTC -3', start
codon in bold and restriction site BamHI underlined.
Primer 107 (SEQ ID NO 6):
[0179] [0180] 5'-CTT CTT AAG TCA ATC AGC GCG GTC TGT GC -3', stop
codon in bold and restriction site AfIII underlined.
Primer 121 (SEQ ID NO 7):
[0180] [0181] 5'-GGG GAT CCA CCA TGA GAT TAT CGA CTT CGA GTC TCT
TCC TTT CCG TGT CTC TGC TGG GGA AGC TGG CCC TCG GGC AAA CTG CAA CTT
CAG CCG C -3', start codon in bold and restriction site BamHI
underlined. Please note that primer 106 maintains the native
bacterial signal sequence, while primer 121 replaces the native
signal with the signal sequence from the fungal amylase SP288 (the
secretion signal from the acid amylase SP288 from A. niger). The
PCR products were digested with restriction enzymes BamHI and AfIII
using standard methods. The resulting fragments were ligated to
BamHI/AfIII digested pEN12516 vector (pENI2516 is described in WO
2004/069872 Example 2) prior to transformation into E. coli DH10B.
Plasmids with the correct sequence, pLAQ055 and pLAQ062, were
selected. The verified constructs were transformed into protoplasts
of Aspergillus oryzae strain ToC1512 (ToC1512 is described in
WO2005/070962, Example 11).
Fermentation
[0182] The transformants were grown in YP medium added 2% maltose
and trace elements for 4-5 days at 34.degree. C. The level of
expression was analysed by standard SDS PAGE analysis, which showed
a strong band of the correct size without any apparent degradation
for both expression strains. The enzymatic activity was analysed
using the EnzCheck.RTM. Paraoxonase Assay Kit (Molecular
Probes.TM., Cat # E33702), which showed high levels of paraoxonase
activity in the growth media of both expression strains. The
protein band was blotted onto a membrane and its N-terminal
sequence was determined using standard methods. Two N-terminal
amino acid sequences were determined: ATQQRTQ and TQQRTQV. The
identity of the protein was thus confirmed and it appears that even
the native signal sequence is processed correctly by A. oryzae. The
result of the determination of the N-terminal demonstrates two
possible positions as the start of the mature enzyme at position 35
and 36 in SEQ ID NO: 2 respectively.
Purification
[0183] Growth medium from a standard fermentation was sterile
filtered using a 0.45 .mu.m membrane filter. The pH of the growth
media was then adjusted to pH 5.8 prior to binding onto a column
with SP-sepharose pre-equilibrated with 50 mM acetate buffer, pH
5.8. The column was then washed with the same buffer before eluting
the protein with a step-wise gradient of NaCl.
Activitv Assay
[0184] The enzymatic activity was determined using the EnzChek.RTM.
Paraoxonase assay from Molecular Probes.TM.. Paraoxonase has
multiple activities including organophosphatase and
phosphotriesterase. The organophosphatase activity confers
protection against toxic organophosphates such as insecticides,
which are a common source of chemical intolerance, and nerve agents
such as sarin and VX. The assay was employed to determine the level
of expression obtained by fermenting the already described
genetically modified organisms. The results clearly indicate that
expression in A. oryzae is superior to all other tested host
organisms. A further advantage is that the enzyme is secreted to
the growth media from this organism.
TABLE-US-00004 Rel. Organism activity Blank 0.00 E. coli,
intracellular 0.25 A. oryzae (native signal), extracellular 18.00
A. oryzae (SP288 signal), extracellular 100.00
[0185] Experiments to identify the cofactor of the enzyme have also
been carried out. Several small scale fermentations were set up,
where only one of the individual components present in the trace
element mixture was added. The fermentation added only Mn resulted
in the highest level of activity, which indicates that Mn is the
preferred cofactor of the enzyme.
TABLE-US-00005 Rel. Added cofactor activity Blank 0 Trace element
mix 53 MgCl.sub.2 11 CaCl.sub.2 5 FeCl.sub.3 8 MnCl.sub.2 100
Sequence CWU 1
1
1111005DNARalstonia pickettii 1atgcgtttgc ttacttctgt cgccgtgata
gccgtggccg ccgcgatggc gacgtctttc 60ggagtcgccc aagcccaaac tgcaacttca
gccgctaccg ctgccacgca acaacgcacc 120caagtgcccg gttactaccg
tatggcgctg ggtgaccacg tggtgaccgc gctctacgac 180ggctacaccg
atctgcccgc caagactctg cttggcctga acgcgcaatc ggtgcagagc
240ctgctggcgc ggatgtttgt atcgaacacg ccgggcatgc agacggcggt
gaacggcttt 300ctgatcgaca cgggtgccaa gcgcatcctc gttgatacgg
ggtcgggcac gtgctttggc 360ccgacgatgg gcggcctggc cggcaacgtc
cgcgcctccg gctatcagcc cgagcagatc 420gatgccgtgc tgctgacgca
cctgcatccg gaccacgcat gcggcctcct gacgccgcaa 480ggtcaaccgg
cctttccgaa tgcgcaggtg tatgtcgccg cgccggaagc cgatttctgg
540ctgagcgaaa cgattgccgc ttccaaaccg agcgacatgc aaccgttctt
caagatggcg 600cgcgactcag tggcgccata tgccgctgcg ggcaagctca
agcaattcaa gccgggtgac 660gaggtggtgc ccggcgtacg gtcggtgcgc
gccaacgggc acacgccggg gcatagcggt 720tacctgttcg catcgaaggg
acacagcctg ctggtgtggg gcgatatcgt gcacagccac 780gccgtgcagt
tccagcaccc cgaggtcgcg ttcgaatatg acgtcgacaa gaaggctgct
840gtcgccacgc gccgcaagct gtttgcggaa gcggcgcgag acaagctctg
ggtggccggc 900gctcatcttc cgttcccggg gctggggcac gtgcgccgcg
agggcaacgc cttcgcctgg 960gtgccgatcg aatacggccc gctgcgcaca
gaccgcgctg attga 10052334PRTRalstonia pickettii 2Met Arg Leu Leu
Thr Ser Val Ala Val Ile Ala Val Ala Ala Ala Met1 5 10 15Ala Thr Ser
Phe Gly Val Ala Gln Ala Gln Thr Ala Thr Ser Ala Ala 20 25 30Thr Ala
Ala Thr Gln Gln Arg Thr Gln Val Pro Gly Tyr Tyr Arg Met 35 40 45Ala
Leu Gly Asp His Val Val Thr Ala Leu Tyr Asp Gly Tyr Thr Asp 50 55
60Leu Pro Ala Lys Thr Leu Leu Gly Leu Asn Ala Gln Ser Val Gln Ser65
70 75 80Leu Leu Ala Arg Met Phe Val Ser Asn Thr Pro Gly Met Gln Thr
Ala 85 90 95Val Asn Gly Phe Leu Ile Asp Thr Gly Ala Lys Arg Ile Leu
Val Asp 100 105 110Thr Gly Ser Gly Thr Cys Phe Gly Pro Thr Met Gly
Gly Leu Ala Gly 115 120 125Asn Val Arg Ala Ser Gly Tyr Gln Pro Glu
Gln Ile Asp Ala Val Leu 130 135 140Leu Thr His Leu His Pro Asp His
Ala Cys Gly Leu Leu Thr Pro Gln145 150 155 160Gly Gln Pro Ala Phe
Pro Asn Ala Gln Val Tyr Val Ala Ala Pro Glu 165 170 175Ala Asp Phe
Trp Leu Ser Glu Thr Ile Ala Ala Ser Lys Pro Ser Asp 180 185 190Met
Gln Pro Phe Phe Lys Met Ala Arg Asp Ser Val Ala Pro Tyr Ala 195 200
205Ala Ala Gly Lys Leu Lys Gln Phe Lys Pro Gly Asp Glu Val Val Pro
210 215 220Gly Val Arg Ser Val Arg Ala Asn Gly His Thr Pro Gly His
Ser Gly225 230 235 240Tyr Leu Phe Ala Ser Lys Gly His Ser Leu Leu
Val Trp Gly Asp Ile 245 250 255Val His Ser His Ala Val Gln Phe Gln
His Pro Glu Val Ala Phe Glu 260 265 270Tyr Asp Val Asp Lys Lys Ala
Ala Val Ala Thr Arg Arg Lys Leu Phe 275 280 285Ala Glu Ala Ala Arg
Asp Lys Leu Trp Val Ala Gly Ala His Leu Pro 290 295 300Phe Pro Gly
Leu Gly His Val Arg Arg Glu Gly Asn Ala Phe Ala Trp305 310 315
320Val Pro Ile Glu Tyr Gly Pro Leu Arg Thr Asp Arg Ala Asp 325
330333DNAArtificialPCR primer 3ggattaatgc gtttgcttac ttctgtcgcc gtg
33430DNAArtificialPCR primer 4ccgctcgaga tcagcgcggt ctgtgcgcag
30533DNAArtificialPCR primer 5gtaggatcca ccatgcgttt gcttacttct gtc
33629DNAArtificialPCR primer 6cttcttaagt caatcagcgc ggtctgtgc
29794DNAArtificialPCR primer 7ggggatccac catgagatta tcgacttcga
gtctcttcct ttccgtgtct ctgctgggga 60agctggccct cgggcaaact gcaacttcag
ccgc 94812PRTArtificialalignment sequence 1 8Ala Cys Met Ser His
Thr Trp Gly Glu Arg Asn Leu1 5 10914PRTArtificialaligment sequence
2 9His Gly Trp Gly Glu Asp Ala Asn Leu Ala Met Asn Pro Ser1 5
10107PRTRalstonia pickettii 10Ala Thr Gln Gln Arg Thr Gln1
5117PRTRalstonia pickettii 11Thr Gln Gln Arg Thr Gln Val1 5
* * * * *
References